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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 俞松良(Sung-Liang Yu) | |
| dc.contributor.author | Chih-Jung Huang | en |
| dc.contributor.author | 黃芝榕 | zh_TW |
| dc.date.accessioned | 2021-06-15T06:13:34Z | - |
| dc.date.available | 2020-08-12 | |
| dc.date.copyright | 2010-09-13 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-08-12 | |
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Barnett Foster, D., et al., Enterohemorrhagic Escherichia coli induces apoptosis which augments bacterial binding and phosphatidylethanolamine exposure on the plasma membrane outer leaflet. Infect Immun, 2000. 68(6): p. 3108-15. 29. Crane, J.K., S. Majumdar, and D.F. Pickhardt, 3rd, Host cell death due to enteropathogenic Escherichia coli has features of apoptosis. Infect Immun, 1999. 67(5): p. 2575-84. 30. Shaw, E., et al., Identification of a novel class in the alpha/beta hydrolase fold superfamily: the N-myc differentiation-related proteins. Proteins, 2002. 47(2): p. 163-8. 31. Lachat, P., et al., Expression of NDRG1, a differentiation-related gene, in human tissues. Histochem Cell Biol, 2002. 118(5): p. 399-408. 32. Kovacevic, Z. and D.R. Richardson, The metastasis suppressor, Ndrg-1: a new ally in the fight against cancer. Carcinogenesis, 2006. 27(12): p. 2355-66. 33. Cangul, H., et al., Enhanced overexpression of an HIF-1/hypoxia-related protein in cancer cells. Environ Health Perspect, 2002. 110 Suppl 5: p. 783-8. 34. Sibold, S., et al., Hypoxia increases cytoplasmic expression of NDRG1, but is insufficient for its membrane localization in human hepatocellular carcinoma. FEBS Lett, 2007. 581(5): p. 989-94. 35. Werth, N., et al., Activation of hypoxia inducible factor 1 is a general phenomenon in infections with human pathogens. PLoS One. 5(7): p. e11576. 36. Greijer, A.E. and E. van der Wall, The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis. J Clin Pathol, 2004. 57(10): p. 1009-14. 37. Chen, D., et al., Direct interactions between HIF-1 alpha and Mdm2 modulate p53 function. J Biol Chem, 2003. 278(16): p. 13595-8. 38. Stein, S., et al., NDRG1 is necessary for p53-dependent apoptosis. J Biol Chem, 2004. 279(47): p. 48930-40. 39. Yan, X., et al., N-Myc down-regulated gene 1 mediates proliferation, invasion, and apoptosis of hepatocellular carcinoma cells. Cancer Lett, 2008. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47702 | - |
| dc.description.abstract | 中文摘要
微核醣核酸(microRNAs)是一群微小的RNA，它透過抑制標的基因的轉譯或降解基因的訊息RNA來調控標的基因的表現。近年來，微核醣核酸於宿主(host)與病原體(pathogen)交互作用的領域當中，漸被發現扮演著重要的角色。為了要了解微核醣核酸是否參與宿主與病原菌之間交互作用的調控，實驗中以出血性大腸桿菌(E. coli O157: H7)感染大腸癌細胞株HT-29，利用檢測乳糖脫氫脢(LDH)的釋放量來代表細胞存活曲線，並根據細胞死亡狀況於感染後收取未感染:0小時以及兩個感染後被認為有意義的時間點，分別為8和12小時的細胞。接著利用微核醣核酸微陣列晶片(microRNA microarray)的平台篩選出可能參與調控的微核醣核酸及以定量反轉錄酶-聚合酶連鎖反應(reverse-transcriptase polymerase chain reaction)進行驗證。實驗結果發現miR-320, miR-345, miR-422a和 miR-331-5p的表現量會隨著大腸桿菌感染時間增加而有減少的情形。另外，我們也同時以基因表現微陣列晶片(gene expression array)的平台進行核醣核酸(mRNA)的分析。分析結果顯示部分基因的表現明顯受到細菌感染所影響，我們推測這些基因產物可能參與在發炎、免疫以及細胞凋亡(apoptosis)相關的路徑當中，實驗結果和已知大腸桿菌感染造成的結果和實驗中的表現型結果一致。之後利用微核醣核酸以及基因表現兩個微陣列平台預測其中一個微核醣核酸可能的標的基因NDRG1。實驗中期望可以證實微核醣核酸可透過抑制標的基因的表現進而影響宿主和病原菌之間的交互作用。 | zh_TW |
| dc.description.abstract | Abstract
MicroRNAs are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Role of microRNAs has been highlighted in pathogen-host interactions recently. To identify cellular microRNAs involved in the host response to E. coli O157: H7 infection, we first assess the viability of E. coli-infected HT-29 cells with LDH release assay at different time points post infection. Then a comprehensive microRNA profiling assay of E. coli O157: H7-infected HT-29 cells was performed by microRNA microarray (TaqMan Low Density Array) and validated with quantitative reverse-transcriptase polymerase chain (qRT-PCR) reaction. Results showed that the level of miR-320, miR-345, miR-422 and miR-331-5p in HT-29 cells was significantly reduced over time after exposure to E. coli O157: H7. In addition, gene expression array analysis revealed that the mRNAs involved in inflammation, immune response, and apoptosis, which might be related to phenotype in our experiment. Next, we predicted the potential targets of miR-422 in which NDRG1, was identified by side-by-side comparison of predicted microRNA target genes and the gene expression array data. Up to our best knowledge, this is the first study on the altered expression of host microRNAs response to E. coli O157: H7 infection. Our findings might support the hypothesis that certain microRNAs are essential in the host-pathogen interactions by targeting essential genes. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T06:13:34Z (GMT). No. of bitstreams: 1 ntu-99-R97424007-1.pdf: 2010344 bytes, checksum: e04407f51db98d505f22b42c8c6ab220 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | Contents
口試委員會審定書…………………………………………………….……………. I 誌謝…………………………………………………………………….…………….II 中文摘要…………………………………………………………………………….III 英文摘要…………………………………………………………………………….IV 1. Introduction…….………………………………………………………….……1 1.1 Introduction of Enterohemorrhagic Escherichia coli serotype O157: H7......2 1.1.1 Escherichia coli…..…………………...………………………………2 1.1.2 Enterohemorrhagic Escherichia coli serotype O157: H7……….…...2 1.1.3 Symptoms of EHEC O157: H7…………………………………..…..3 1.1.4 Virulent factors of EHEC O157: H7…………………………………4 1.2 Host in response to infection ………………………..………………..…… .4 1.3 Whole transcriptome analysis of HT-29 cells ……………………………….6 1.4 MicroRNAs…………………………………………….………….…………6 1.5 Aims of the study……………………………………...………………..……8 2. Materials and Methods…..…………………….……….…………………..... 9 2.1 Bacteria and culture condition ……...………………………………..……..10 2.2 Cell culture………………………………………………………….………10 2.3 In vitro infection assay………………………………..…………..………...10 2.4 Adhesion assay……...………………………..……………………………..11 2.5 Cell adhesion assay…………………………………...…………………….12 2.6 LDH release assay………………………………………….…...…………..12 2.7 Immunofluorescence staining………………………………….……………13 2.8 RNA isolation………………………………………………………………13 2.9 MicroRNA microarray profiling…………………………….……………...14 2.10 Gene expression microarray profiling……………………………………..15 2.11 Quantification of microRNAs with real-time PCR……………………......15 2.12 Quantitative real-time PCR for transcript expression………………..16 2.13 Construction of microRNAs expressing plasmids………………………...17 2.14 Luciferase assay………………………………………….………………..18 3. Results……………………………………………….………………………….19 3.1 Select for a suitable cell lines ………………………………………………20 3.2 Optimization of Infection condition……………………….………………..20 3.2.1 Characterizing the lysis efficiency of Triton X-100…………………20 3.2.2 Optimization of the multiplicity of infection……………..…………21 3.2.3 Infection rate of E. coli O157: H7 test by Immunofluorescence…….21 3.3 Time course of infection assay and phenotypes searching...………………..22 3.3.1 Time course of adhesion assay...………………………………….…22 3.3.2 Time course of actin rearrangement assay by Immuno- fluorescence…………………………………………………………23 3.3.3 Time course of cell adhesion assay…………………………………23 3.3.4 Time course of LDH release assay...…...….………………………...24 3.4 E. coli O157: H7 alters microRNAs levels in HT-29 host cells...………......25 3.5 Confirmation of differentially expressed microRNAs ...…………...………26 3.6 Global mRNA transcription profiles of HT-29 cells ………………….……28 3.7 Confirmation of differentially expressed mRNAs……………….…………29 3.8 Correlation of the alterated microRNAs and mRNA expression during E. coli O157: H7 infection……………………………………………….29 4. Discussion………………………………………………………..……..……….31 4.1 Phenotype selection of E. coli O157: H7 infection to HT-29 cell………….32 4.2 Selected Potential microRNAs and microRNAs in host defense……...……34 4.3 Host cell signaling responses…………………………………………..…...34 4.4 Host response and apoptosis……………………………………………..…35 4.5 NDRG1 and cell death……………………………………………………...36 4.6 Future perspectives………………………………………………..………...37 5. References..………………………………………………………….………...39 6. Figures……………………………………………………..…………..………..46 Figure 1. Optimization of the concentration of TritonX-100………………..…47 Figure 2. Optimization of the multiplicity of infection.………...……....….…...48 Figure 3. Adherence of E. coli O157: H7 to HT-29 cells….……………………49 Figure 4. Time course of adhesion assay.………...……………………………51 Figure 5. Actin rearrangement observed by fluorescence microscope.…………52 Figure 6. Time course of cell adhesion assay...…………………………………54 Figure 7. Time course of LDH release assay..………………………………….56 Figure 8. Altered expression of selected microRNAs confirmed by qRT-PCR...62 Figure 9. Down-regulation of miR-320, miR-345, miR-422a, miR-331-5p in response to E. coli O157: H7 infection…………………………..64 Figure 10. Numbers of differentially regulated genes in HT-29 cells after 12 hr post infection of E. coli O157: H7……….…..….…………..……...66 Figure 11. Analysis of the expression levels of NDRG1 and CDKN1A……......67 7. Tables………………………………………………………………...……….…68 Table 1. All primers used in the studies…..….……………………………….....69 Table 2. Differential gene expression between experimental and control (fold change > 2x) at 12 hr were analyzed by GeneGo process network , one of the analysis tool in MetaCore software………...…71 8. Supplementary Figures……………………………..………………………….73 Supplementary Figure 1. Vector used in the studies.………...…………………74 Supplementary Figure 2.The quality of the RNA was verified using an Agilent 2100 Bioanalyzer performed by RNA integrity number……...……….75 Supplementary Figure 3. A sequence alignment of miR-422a and its target sites in coding region of NDRG1………………………….….76 | |
| dc.language.iso | en | |
| dc.title | 探討微核醣核酸於大腸桿菌O157: H7引發細胞死亡所扮演的角色 | zh_TW |
| dc.title | Characterizing the role of microRNAs in
E. coli O157: H7 induced cell death | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鄧麗珍,廖淑貞,顏伯勳 | |
| dc.subject.keyword | 病原體-宿主交互作用,出血性大腸桿菌O157: H7,微核醣核酸,細胞死亡,NDRG1, | zh_TW |
| dc.subject.keyword | pathogen-host interaction,Enterohemorrhagic Escherichia coli serotype O157: H7,microRNAs,cell death,NDRG1, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2010-08-12 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
| Appears in Collections: | 醫學檢驗暨生物技術學系 | |
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